Article pubs.acs.org/jnp
Semisynthesis of the Antiviral Abietane Diterpenoid Jiadifenoic Acid C from Callitrisic Acid (4-Epidehydroabietic Acid) Isolated from Sandarac Resin Miguel A. González* and Ramón J. Zaragozá Departamento de Química Orgánica, Universidad de Valencia, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain S Supporting Information *
ABSTRACT: The semisynthesis of the antiviral abietane diterpenoid (+)-jiadifenoic acid C starting from the available methyl ester of callitrisic acid (4-epidehydroabietic acid) isolated from sandarac resin is reported. A protocol for the isolation of methyl callitrisate (methyl 4-epidehydroabietate) in gram quantities from sandarac resin is also described. Allylic C-17 oxygenation was introduced by regioselective dehydrogenation of the isopropyl group of methyl callitrisate with DDQ followed by seleniumcatalyzed allylic oxidation. Ester hydrolysis afforded (+)-jiadifenoic acid C in 22% overall yield from methyl callitrisate. This semisynthetic route provides a convenient source of this anti-Coxsackie virus B natural product for further biological studies.
A
bietane diterpenoids are widely distributed natural products in the plant kingdom with a wide range of biological activities.1 The recent isolation in 2013 of the abietane-type diterpenoids jiadifenoic acids C (1) and B (2), together with the known 7α-hydroxycallitrisic acid (3) (Figure 1) and related metabolites, has led to some biological studies aimed at their antiviral properties by Yu and co-workers.2,3 Figure 2. Compounds isolated from Sandarac resin.
methyl callitrisate (methyl 4-epidehydroabietate) (7) in gram quantities from sandarac resin is described. Therefore, the synthetic strategy provides an efficient alternative to obtain (+)-jiadifenoic acid C (1) for additional studies.
■
Figure 1. Compounds isolated from lllicium jiadifengpi.
RESULTS AND DISCUSSION Methyl callitrisate (7) was selected as starting material for the semisynthesis of jiadifenoic acid C (1), since it contains the aromatic ring and the C-19 carboxylic function present in the target molecule. Methyl callitrisate 7 could be obtained semisynthetically from commercial (−)-abietic acid via its conversion into dehydroabietic acid5 and subsequent conversion into (+)-callitrisic acid6 followed by esterification. It can also be obtained from the resin of the white cypress pine, Callitris columellaris, involving a problematic chromatographic separation including expensive silver nitrate.7 The isolation of callitrisic acid (4) from Australian sandarac resin was reported without experimental data. A chemical modification of the accompanying pimaric-type acids obtained during the isolation was used to facilitate the separation of products.8
These compounds have proved to be potent inhibitors of some enteroviruses, in particular, the Coxsackie viruses B, which are reported to be associated with the development of myocarditis, pancreatitis, meningitis, and encephalitis.4 Compounds 1−3 occur in minute amounts in the roots of the medium-sized tree Illicium jiadifengpi B.N. Chang, which is primarily distributed in southern China.2 Its bark has been used as a Traditional Chinese Medicine for the treatment of rheumatism. The limited access to these compounds including time-consuming isolation protocols has prompted a study of alternative sources of these materials for further development as antiviral drugs. In this paper, a straightforward semisynthetic sequence (Scheme 1) to jiadifenoic acid C (1) from the available methyl ester (7) obtained by esterification of callitrisic acid (4epidehydroabietic acid) (4) isolated from Moroccan sandarac resin (Figure 2) is reported. A protocol for the production of © XXXX American Chemical Society and American Society of Pharmacognosy
Received: July 15, 2014
A
dx.doi.org/10.1021/np500569y | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Scheme 1. Semisynthesis of Jiadifenoic Acid C from Methyl Callitrisate (7)
Thus, heating a solution of methyl callitrisate (7) and DDQ under reflux in benzene gave a mixture, inseparable by column chromatography, of starting material (7) (40%) and alkene 8 (60%) in 79% yield (ratio calculated by integration of 1H NMR signals; 6.89 ppm, broad singlet for methyl callitrisate; 5.31 ppm, singlet for the alkene). Although the yield of alkene 8 is moderate (45%), the fact that unconsumed methyl callitrisate (7) can be recovered in the next step and may be efficiently recycled makes this reaction a useful method for the regioselective functionalization of the C-13 isopropyl group. Attempts to improve this reaction with other solvents were unsuccessful. The next step was the introduction of the hydroxy group. An allylic oxidation using SeO2 would afford the required allylic alcohol. Thus, SeO2-catalyzed (0.15 equiv) oxidation of a mixture of 7/8 (ca. 40:60) with tert-butyl hydroperoxide (TBHP) as co-oxidant (1.5 equiv), in HOAc and DCM as solvent at room temperature during 20 h,14 occurred selectively at C-17, affording the allylic alcohol 9 (50% yield). The alcohol 9 was accompanied by the corresponding unsaturated aldehyde 10 (12%) and unreacted alkene 8 (22%). The aldehyde 10 and unreacted alkene 8 were difficult to separate by column chromatography. In order to optimize this reaction, the number of equivalents of TBHP was increased to 2.3, resulting in a crude mixture of alcohol 9 (57%), aldehyde 10 (14%), and unreacted alkene 8 (18%). Attempts to complete this reaction with higher amounts of co-oxidant and reaction time were unsuccessful. For convenience of product separation and to increase the yield of alcohol 9, the mixture containing 14% of aldehyde was subjected to reduction with sodium borohydride (NaBH4) in a mixture of MeOH−DCM. This lead to the allylic alcohol 9 in 65% overall yield for the allylic oxidation and reduction steps. An alternative route to obtain alcohol 9 by epoxidation of 8 with m-chloroperbenzoic acid and base-promoted isomerization15 of the corresponding epoxide was also studied, but it gave lower yields. Next, the hydrolysis of the ester group in 9 to afford jiadifenoic acid C (1) was studied. First attempts by treatment of 9 with potassium tert-butoxide in DMSO gave poor yields (ca. 30% yield). However, treatment of 9 with LiI in refluxing collidine, followed by chromatography, afforded (+)-jiadifenoic acid C (1) in 75% yield. Its 1H and 13C NMR spectra, together with HRMS data (calcd for C20H27O3 [M + H]: 315.1960; found: 315.1963) and specific rotation data ([α]20D = +129, c 0.6, MeOH), corresponded to those reported for the natural
Herein, a chemical modification strategy to separate a mixture of the methyl esters of sandaracopimaric acid (5) and isopimaric acid (6) isolated together with methyl callitrisate (7) from Moroccan sandarac resin (Figure 2) is described. Methyl callitrisate (7) was isolated from an available sample of Moroccan sandarac resin following the method of Carman and Deeth.7 This resulted in a ca. 30% weight of crude resin acids with identical retention factors (Rf) by TLC. The NMR data of this mixture of acids showed that the major components were callitrisic acid (4) and sandaracopimaric acid (5) (ca. 1:1 ratio) along with a minor component. A pimaric-type diterpene was suspected as the minor component of the mixture of acids since in the 1H NMR spectrum similar signals in addition to the signals due to sandaracopimaric acid were observed. In the 13C NMR spectrum of the crude acids there were some minor signals at 150.2 (doublet), 135.5 (singlet), 120.9 (doublet), and 109.2 ppm (triplet) reminiscent of a pimaric-type diterpenoid. A search by chemical shift in the NAPROC-13 database9 suggested that these signals were assignable to those of isopimaric acid (6).10 The integration of 1H NMR signals (6.88 ppm, singlet for callitrisic acid; 5.32 ppm, doublet for isopimaric acid; and 5.22 ppm, singlet for sandaracopimaric acid) indicates that the minor component of the mixture was present in ca. 10−12%. Therefore, the content in callitrisic acid (4) and sandaracopimaric acid (5) in the sandarac resin was ca. 12− 13% each.11 Next, the crude acids were methylated in quantitative yield by treatment with LiOH and MeI. The resulting esters showed identical Rf values by TLC, hence complicating their chromatographic separation. A selective procedure for modification of the vinyl group present in the pimaric-type esters was used in order to increase the polarity of those compounds. Thus, the mixture of esters was subjected to an oxymercuration−reduction reaction12 (70% yield), which allowed the ready separation of unreacted methyl callitrisate (7). With methyl callitrisate (7) in hand, the focus was shifted to the synthetic target, compound 1 (jiadifenoic acid C). This aromatic diterpenoid shows interesting structural features such as an allylic alcohol moiety, as well as a C-19 carboxylic group. It is known that methyl callitrisate (7) can be regioselectively dehydrogenated by treatment with 2,3-dichloro-5,6-dicyanoquinone (DDQ) to give the corresponding alkene 8 (methyl abieta-8,11,13,15-tetraen-19-oate),13 which contains the isopropenyl moiety present in jiadifenoic acid C (1) (Scheme 1). B
dx.doi.org/10.1021/np500569y | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Synthesis. Methyl Abieta-8,11,13,15-tetraen-19-oate (8). This compound was prepared following the procedure of Abad and coworkers13 but using 30% less solvent. Methyl (+)-17-Hydroxyabieta-8,11,13,15-tetraen-19-oate (9, methyl jiadifenoate C). To a mixture of alkene 8 (60% purity, 400 mg, 0.77 mmol), SeO2 (13 mg, 0.11 mmol), and HOAc (6 μL, 0.11 mmol) in DCM (0.8 mL) was added tert-butyl hydroperoxide (70%, 0.23 mL, 1.7 mmol), and the mixture stirred for 18 h. The reaction mixture was diluted with EtOAc, washed with brine, dried, and concentrated. The resulting mixture (light orange oil) was reduced with NaBH4 (15 mg) in MeOH (1 mL) and DCM (0.5 mL) at 0 °C. After 30 min, the mixture was allowed to warm to rt, and stirring continued for an additional 45 min. The mixture was concentrated, dissolved in EtOAc (15 mL), washed with brine, dried, and concentrated. The residue was chromatographed on silica eluting with n-hexane−EtOAc (7:3) to give 190 mg of methyl callitrisate (7) containing alkene 8 as impurity and 165 mg (65%) of compound 9 as a colorless oil: [α]20D +120 (c 0.5, CHCl3); 1H NMR (300 MHz) δ 7.26−7.23 (2H, m), 7.14 (1H, s), 5.44 (1H, s), 5.30 (1H, s), 4.52 (2H, s), 3.67 (3H, s), 2.90−2.75 (2H, m), 1.29 (3H, s), 1.04 (3H, s); 13C NMR (75 MHz) δC 177.8 (s), 147.9 (s), 146.9 (s), 135.4 (s) × 2, 126.5 (d), 125.8 (d), 123.5 (d), 111.8 (t), 64.9 (t), 52.7 (d), 51.2 (q), 43.9 (s), 39.2 (t), 38.3 (s), 37.6 (t), 32.1 (t), 28.5 (q), 22.8 (q), 20.9 (t), 19.9 (t); HRMS (ESI) m/z 329.2125 [M + H]+, calcd for C21H29O3 329.2117. Jiadifenoic Acid C (1). A solution of ester 9 (145 mg, 0.44 mmol) in 2,4,6-collidine (2 mL) was treated with LiI (413 mg, 3.1 mmol) and heated at reflux for 2 h. The reaction mixture was cooled to rt, poured into 6 N HCl (15 mL), and extracted with DCM (3 × 10 mL). The combined organic extracts were washed with brine, dried, and concentrated. The resulting brown residue was chromatographed on silica eluting with n-hexane−EtOAc (3:7) to give 104 mg of jiadifenoic acid C (1) as a white, microcrystalline solid: mp 177−179 °C; [α]20D +129 (c 0.6, MeOH); 1H NMR (methanol-d4, 300 MHz) δ 7.22−7.18 (2H, m), 7.09 (1H, s), 5.38 (1H, s), 5.27 (1H, s), 4.41 (2H, s), 2.90− 2.60 (2H, m), 1.28 (3H, s), 1.12 (3H, s); 13C NMR (methanol-d4, 75 MHz) δC 181.4 (s), 149.0 (s), 148.8 (s), 137.2 (s), 136.3 (s), 127.5 (d), 126.6 (d), 124.6 (d), 111.4 (t), 64.8 (t), 54.2 (d), 44.8 (s), 40.6 (t), 39.6 (s), 38.7 (t), 33.1 (t), 29.2 (q), 23.7 (q), 22.3 (t), 21.1 (t); HRMS (ESI) m/z 315.1963 [M + H]+, calcd for C20H27O3 315.1960.
product (naturally derived jiadifenoic acid C had [α]D = +135.5 (c 0.074, MeOH)). In summary, the natural product (+)-jiadifenoic acid C (1), an anti-Coxsackie virus B agent, was synthesized from available callitrisic acid (4) isolated from sandarac resin, in 22% overall yield. This synthesis solves the problem of availability of the diterpenoid for potential biomedical applications, as it is an excellent starting material for the synthesis of other potential bioactive molecules.
■
EXPERIMENTAL SECTION
General Experimental Procedures. The melting points were measured with a Büchi 535 apparatus and are uncorrected. Optical rotations were measured using a 5 cm cell in a Schmidt-Haensch Polartronic-D polarimeter. NMR spectra were recorded on a 300 MHz spectrometer. All spectra were recorded in CDCl3 as solvent unless otherwise stated. Complete assignments of 13C NMR multiplicities were made on the basis of DEPT experiments. J values are given in Hz. MS data were acquired on a QTOF spectrometer. Reactions were monitored by TLC using Merck silica gel 60 F-254 in 0.25 mm thick plates. Compounds on TLC plates were detected under UV light at 254 nm and visualized by immersion in a 10% sulfuric acid solution and heating with a heat gun. Purifications were performed by flash chromatography on Merck silica gel (230−400 mesh). Commercial reagent grade solvents and chemicals were used as purchased unless otherwise noted. Combined organic extracts were washed with brine, dried over anhydrous NaSO4, filtered, and concentrated under reduced pressure. Isolation of Methyl Callitrisate (7). Callitrisic acid (4) was isolated from sandarac resin7 together with sandaracopimaric acid (5) and isopimaric acid (6). Thus, 50 g of resin was dissolved in 250 mL of Et2O. The liquid was decanted and extracted with aqueous Na2CO3 (30 g in 250 mL in three portions). The combined aqueous phase was acidified to pH = 1 (ca. 110 mL of 6 M HCl) and extracted with Et2O (3 × 100 mL). The combined organic extracts were washed with brine, dried, and concentrated to give 15 g of crude acids as an orange oil. Crude acids (15 g) were dissolved in DMF (110 mL), and LiOH· H2O (6.3 g, 0.15 mol) was added. The mixture was stirred for 20 h at room temperature and cooled to 0 °C before adding MeI (19 mL, 0.3 mol). The mixture was allowed to warm to room temperature (rt) and stirring continued for 20 h. The reaction mixture was poured into H2O (300 mL), extracted with n-hexane (4 × 125 mL), washed with brine, dried, and concentrated to give the crude esters (15.2 g). The separation of esters (7.6 g) was achieved by chemical modification of methyl pimarates by treatment with Hg(OAc)2 (5.8 g) in THF (18 mL) and H2O (6 mL) and 125 μL of 70% HClO4. After being stirred for 2 h, the mixture was cooled to 0 °C, and 10% NaOH (15 mL) was added, followed by the dropwise addition of a solution of NaBH4 (600 mg) in 10% NaOH (5 mL). The mixture was allowed to warm to rt and stirred for 2 h. After cooling to 0 °C, the reaction was quenched with 1 N HCl (ca. 35 mL) until no more effervescence could be detected, H2O (100 mL) was added, and the mixture was extracted with Et2O (3 × 125 mL). The combined extracts were washed with saturated NaHCO3 and brine, dried, and concentrated. Finally, column chromatography of the oily residue eluting with n-hexane−EtOAc (8:2 to 6:4) gave unchanged methyl callitrisate (7, 2.8 g, ca. 11% yield from the resin) as a colorless oil that solidified upon standing, followed by the corresponding epimeric alcohols12a (3.4 g) (Supporting Information). Methyl callitrisate (7): mp 79−80 °C (lit.,8 80.5−81 °C); [α]20D +135 (c 0.5, EtOH) (lit.,8 +136, c 1); 1H NMR (300 MHz) δ 7.19 (1H, d, J = 6), 7.00 (1H, dd, J = 6, 3), 6.89 (1H, br s), 3.66 (3H, s), 2.90−2.75 (3H, m), 1.27 (3H, s), 1.23 (6H, d, J = 6), 1.03 (3H, s); 13C NMR (75 MHz) δC 178.0 (s), 145.6 (s), 145.4 (s), 135.0 (s), 126.8 (d), 125.5 (d), 124.0 (d), 52.9 (d), 51.2 (q), 43.9 (s), 39.3 (t), 38.1 (s), 37.7 (t), 33.4 (d), 32.1 (t), 28.5 (q), 23.9 (q) × 2, 23.0 (q), 21.0 (t), 19.9 (t); HRMS (ESI) m/z 315.2332 [M + H]+, calcd for C21H31O2 315.2324.
■
ASSOCIATED CONTENT
S Supporting Information *
Copies of 1H NMR and 13C NMR spectra for all relevant compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: +34 96 3544328. Tel: +34 96 3543880. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS Financial support from the Spanish Ministry of Science and Education, under a “Ramón y Cajal” research grant, and also from the Generalitat Valenciana is gratefully acknowledged.
■
REFERENCES
(1) For selected reviews on this topic, see: Hanson, J. R. Nat. Prod. Rep. 2013, 30, 1346−1356 and preceding contributions of the author. (2) (a) Zhang, G.-J.; Li, Y.-H.; Jiang, J.-D.; Yu, S.-S.; Qu, J.; Ma, S.-G.; Liu, Y.-B.; Yu, D.-Q. Tetrahedron 2013, 69, 1017−1023. (b) Wang, Y.D.; Zhang, G.-J.; Qu, J.; Li, Y.-H.; Jiang, J.-D.; Liu, Y.-B.; Ma, S.-G.; Li, Y.; Lv, H.-N.; Yu, S.-S. J. Nat. Prod. 2013, 76, 1976−1983. (c) Fang, Z.-F.; Zhang, G.-J.; Chen, H.; Bai, J.; Yu, S.-S.; Liu, Y.; Wang, W.-J.;
C
dx.doi.org/10.1021/np500569y | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Ma, S.-G.; Qu, J.; Xu, S. Planta Med. 2013, 79, 142−149. (d) Zhang, G.-J.; Li, Y.-H.; Jiang, J.-D.; Yu, S.-S.; Wang, X.-J.; Zhuang, P.-Y.; Zhang, Y.; Qu, J.; Ma, S.-G.; Li, Y.; Liu, Y.-B.; Yu, D.-Q. Tetrahedron 2014, 70, 4494−4499. (3) Diterpene acid for preventing Coxsackie virus, preparation method, pharmaceutical composition, and applications thereof, CN 103450011 A. (4) Rotbart, H. A. Antiviral Res. 2002, 53, 83−89. (5) González, M. A.; Pérez-Guaita, D.; Correa-Royero, J.; Zapata, B.; Agudelo, L.; Mesa-Arango, A.; Betancur-Galvis, L. Eur. J. Med. Chem. 2010, 45, 811−813. (6) Pelletier, S. W.; Herald, D. L. Chem. Commun. 1971, 10−11. (7) Carman, R. M.; Deeth, H. C. Aust. J. Chem. 1967, 20, 2789−2793. (8) Gough, L. J. Tetrahedron Lett. 1968, 3, 295−298. (9) López-Pérez, J. L.; Therón, R.; del Olmo, E.; Díaz, D. Bioinformatics 2007, 23, 3256−3257. (10) Ryu, Y. B.; Jeong, H. J.; Kim, J. H.; Kim, Y. M.; Park, J.-Y.; Kim, D.; Naguyen, T. T. H.; Park, S.-J.; Chang, J. S.; Park, K. H.; Rho, M.C.; Lee, W. S. Bioorg. Med. Chem. 2010, 18, 7940−7947. (11) Sugimoto, N.; Kuroyanagi, M.; Kato, T.; Sato, K.; Tada, A.; Yamazaki, T.; Tanamoto, K. J. Food Hyg. Soc. Jpn. 2006, 47, 76−79. (12) (a) Herz, W.; Hall, A. L. J. Org. Chem. 1974, 39, 14−20. (b) Abad, A.; Agulló, C.; Arnó, M.; Domingo, L. R.; Zaragozá, R. J. J. Org. Chem. 1990, 55, 2369−2373. (13) Abad, A.; Arnó, M.; Peiró, M.; Zaragozá, R. J. Tetrahedron 1991, 47, 3829−3844. (14) Lempers, H. E. B.; Ripollès i Garcia, A.; Sheldon, R. A. J. Org. Chem. 1998, 63, 1408−1413. (15) Crandall, J. K.; Apparu, M. Org. React. 1983, 29, 345−443.
D
dx.doi.org/10.1021/np500569y | J. Nat. Prod. XXXX, XXX, XXX−XXX
